High risk human papillomavirus (HPV) causes cervical dysplasia which can progress to cervical cancer, the second leading cause of cancer-related deaths among women. Therapeutic treatments for cervical cancer are highly toxic and often negatively impact quality of life. Cervical cancer is prevented by detection and destruction of cervical dysplasia involving significant cost, discomfort and potential loss of fertility, althogh the majority of these lesions will not progress to cancer. We developed a non-toxic cancer drug that has a mechanistic profile suitable for application in cervical dysplasia and cancer. Our drug, called SHetA2 (NSC 721689) counteracts the cell cycle regulatory consequences of high risk HPV. Extensive preclinical studies of SHetA2 demonstrated cancer therapeutic and chemoprevention activity in vitro and in vivo, lack of mutagenicity or teratogenicity, no toxicity and a pharmacologic profile suitable for an oral therapeutic agent. A pre-Investigational New Drug (IND) meeting report from the US Food and Drug Administration (FDA) listed no obstacles for our moving forward to a Phase 0 Clinical trial of SHetA2 in cervical cancer. In this project, w hypothesize that SHetA2 causes G1 arrest and apoptosis in human and murine cervical tumors by counteracting the alterations in cyclin D1 and other cell cycle regulatory proteins caused by HPV. We will test this hypothesis by conducting a Phase 0 clinical trial in Stage IB2-IVB cervical cancer patients to determine if oral administration of SHetA2 capsules can achieve systemic and cervical tissue concentrations of SHetA2 known to regulate cyclins A, D1 and E, and RB phosphorylation events. Pre- and post-treatment cervical tumor biopsies and a time course of collected peripheral blood mononuclear cells (PBMCs) collected in this trial will be used to determine which Rb pathway defects found in cervical cancer are altered by SHetA2 treatment. In addition, we will compare oral capsule and vaginal suppository SHetA2 formulations for alterations of cell cycle regulatory proteins in association with reduction of cervical tumor formation in K14-HPV16 transgenic mice. Cell cycle proteins, such as cyclins A, D and E, that are counter- regulated by HPV and SHetA2 will be tested for their direct involvement in SHetA2 regulation of G1 cell cycle arrest and apoptosis. These studies will lay the groundwork for future clinical trials in cervical dysplasia and cancer by determining the pharmacokinetics (PK) and pharmacodynamics (PD) of SHetA2, and by determining whether oral or vaginal delivery will achieve higher tissue concentrations of SHetA2, greater alterations in cell cycle proteins and greater prevention of cervical cancer in HPV transgenic mice. Our mechanistic studies will develop candidate PD biomarkers for use in future clinical trials planned for cervical dysplasia and will provide increased knowledge of HPV associated cell cycle defects in cervical dysplasia and cancer.